Evaluation of the Indazole Analogs of 5-MeO-DMT and Related Tryptamines as Serotonin Receptor 2 Agonists

Herein, we report the synthesis and characterization of a novel set of substituted indazole-ethanamines and indazole-tetrahydropyridines as potent serotonin receptor subtype 2 (5-HT2) agonists. Specifically, we examine the 5-HT2 pharmacology of the direct indazole analogs of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and related serotonergic tryptamines, and highlight the need for rigorous characterization of 5-HT2 subtype selectivity for these analogs, particularly for the 5-HT2B receptor subtype. Within this series, the potent analog VU6067416 (19d) was optimized to have suitable preclinical pharmacokinetic properties for in vivo dosing, although potent 5-HT2B agonist activity precluded further characterization for this series. Additionally, in silico docking studies suggest that the high potency of 19d may be a consequence of a halogen-bonding interaction with Phe2345.38 in the 5-HT2A orthosteric pocket.

−6 Indeed, a number of encouraging clinical reports, highlighting the efficacy of these and related psychedelics, have begun to emerge in the literature, and as of 2021 at least 70 registered clinical studies using psychedelics have been reported. 4Results of a phase 3 clinical study evaluating 3,4-methylenedioxymethamphetamine (MDMA), a phenethylamine in the entactogen class of psychedelics, 7 were recently published and demonstrated that the compound was a safe and effective treatment for severe symptoms observed in PTSD patients. 8n addition to DMT and 5-MeO-DMT, the tryptamine class of psychedelics includes psilocybin (a phosphate ester prodrug of the active 5-HT 2A agonist psilocin) 9 and LSD (a semisynthetic ergoline alkaloid), 10 and the first specific associations of tryptamine structures with psychedelic experiences were first described in the literature at least as early as the 1940s. 6,11,12Given the long history of this class of compounds and the number of recent reports detailing various tryptamine analogs as novel 5-HT 2A agonists, 13−17 it is therefore perhaps surprising that very few reports exist of a direct indazole analog of a serotonergic-type tryptamine.The first such report, published in 1957, describes a synthetic method by which to access the direct 1H-indazole analog of tryptamine itself (3, see Figure 1), although no pharmacology data are reported. 18In keeping with the recent resurgence in psychedelic research, this manuscript remained one of the only reports describing an indazole-substituted serotonergic until very recently, when a selection of patent applications emerged in the literature which describe a wide variety of N-substituted tryptamines and tryptamine isosteres 19,20 (including the direct 1H-indazole analog of 5-MeO-DMT). 20ncorporation of the indazole motif has yielded compounds with excellent properties across a number of drug discovery programs 21−25 and is present in no less than 43 compounds undergoing clinical evaluation (as of 2021). 26−29 We were therefore also interested in examining the indazole isosteres of classical psychedelics (including 5-MeO-DMT) to ultimately (1) profile the 5-HT 2A potency and serotoninsubtype selectivity of 2-aza-5-MeO-DMT and compare our data to the known literature (with a parallel emphasis on the generation of novel analogs) and (2) understand the overall properties of an indazole series of tryptamines with respect to preclinical pharmacokinetics (PK).
The direct 1H-indazole analog of 5-MeO-DMT, compound 6a, was accessed as shown in Scheme 1. Tertiary amides 5a and 5b were generated from commercially available methyl ester 4 via hydrolysis and amide coupling, followed by reduction with either lithium aluminum hydride or lithium aluminum deuteride to generate amines 6a−6c.1-Methylindazole analog 11 was synthesized as shown in Scheme 2. Briefly, triple alkylation of carboxylic acid 7, followed by ester reduction, oxidation, and reductive amination gave 11 (attempts to generate 1H-indazoles 6a−6c using a similar sequence were unsuccessful owing to the apparent instability of indazole aldehydes similar to 10 but lacking the 1-methyl substitution).5-Chloro-and 5-hydroxyindazoles (14 and 16, respectively) were generated using a similar amide reduction sequence to 6a−6c, as shown in Scheme 3.All analogs were then assessed for functional potency across all 5-HT 2 subtypes (Table 1).
In our hands, compound 6a (the direct 1H-indazole analog of 5-MeO-DMT) was found to have low micromolar activity for 5-HT 2A , with higher potency at 5-HT 2B and 5-HT 2C (and was less potent than the indole parent compound across all 5-HT 2 subtypes).1-Methyl analog 11 was markedly less potent at 5-HT 2A compared to both 5-MeO-DMT and 6a.gem-Deutero analog 6b was approximately equipotent at 5-HT 2A relative to its proteo-counterpart 6a.(Recently, this type of deuterium incorporation was found to increase the in vitro stability for a series of DMT analogs in human hepatocyctes. 16n the case of the present indazole analogs, 6b was found to have only marginally lower predicted clearance in human hepatic microsomes compared to 6a (human CL hep = 11.7 and 12.1 (mL/min)/kg, respectively), although a more complete metabolic picture (the effect of deuterium on MAO-mediated oxidation, etc.) would likely be obtained using hepatocytes.)Diethylamine 6c and 5-chloroindazole 14 displayed no appreciable 5-HT 2A functional activity up to 10 μM, whereas 5-hydroxy analog 16 displayed similar potency to 6a for 5-HT 2A (with higher potency for the other subtypes).Within this set, the relatively higher potency observed for 6a and 16 align with the available published data for the corresponding tryptamines, in which a 5-MeO or 5-OH substitution in the  context of the N,N-dimethylamine motif (5-MeO-DMT and bufotenin, respectively) are among the most potent tryptamines described in the literature. 30,31Because the more potent analogs in the present series do not show appreciable selectivity for 5-HT 2A relative to 5-HT 2B and 5-HT 2C , and in fact are largely 5-HT 2B -preferring, this appreciable 5-HT 2B agonist activity may elicit problematic cardiotoxicities for these and related tryptamines. 32−35 Recently, additional chemotypes with some degree of 5-HT 2A subtype selectivity have started to emerge in the literature, and an allosteric approach may prove fruitful toward this end. 19,20,36nterestingly, the direct 1H-indazole analog of 5-MeO-DMT, compound 6a, was previously described to be moderately potent for 5-HT 2A (5-HT 2A EC 50 = 203 nM, E max = 70%), with high selectivity relative to 5-HT 2B (EC 50 > 10 μM) and, to a lesser extent, 5-HT 2C (EC 50 = 532 nM, E max = 72%). 20This large discrepancy between reports for the 5-HT 2B subtype selectivity profile for this compound is noteworthy (483 nM in our hands vs >10 μM), given that agonist activity (nor-dexfenfluramine and related clinical 5-HT 2B agonists) at this receptor presents a well-validated risk for cardiotoxicity. 32lthough it is possible that differences in cell line background and/or receptor expression levels may partially account for this disparate selectivity data (see Supporting Information), this finding highlights the need for rigorous characterization of 5-HT 2A agonists (both known and novel) across multiple cell backgrounds and functional readouts if such compounds are to be safely profiled in the clinic.
In contrast to the acyclic series (Table 1), 1H-indazole 19a and its 1-methyl counterpart 19b were found to be equipotent with respect to 5-HT 2A , with both displaying approximately 10fold higher agonist potency for 5-HT 2B and 5-HT 2C (2methylindazole 19c was found to be inactive at 5-HT 2A up to 10 μM, indicating the importance of the spatial arrangement of the methyl group on the indazole scaffold).All substitutions Table 2.In Vitro Functional Potency for Compounds 19a−f and 20−22 a a Calcium mobilization assays using human 5-HT 2A -CHO, 5-HT 2B -HEK293, and 5-HT 2C -CHO cells.Data represent (n = 1 to 3) independent experiments performed in duplicate (data are ±SEM).See Supporting Information for additional details.ND = not determined.examined at the 5-position, with the exception of 5-phenyl analog 21, were found to be tolerated (19d−f), with 5-bromo analog 19d in particular showing high agonist potency across all 5-HT 2 subtypes (189 nM for 5-HT 2A ).Ring reduction to give saturated piperidine analog 20 was not tolerated, indicating the necessity of the olefin constraint for 5-HT 2A activity.During the preparation of this manuscript, a related patent application containing a series of tetrahydropyridinelinked indoles and indazoles was disclosed, in which additional SAR around this type of 5-HT 2A scaffold is detailed. 19Within this report, the structures and functional 5-HT 2A potency data for 19a and 19f are reported, and interestingly, each compound appears significantly more potent compared to our findings (5-HT 2A EC 50 values of 9.9 and 17.8 nM, respectively), although no selectivity relative to 5-HT 2B and 5-HT 2C is reported for these analogs.In our hands, 19a and 19f appear to be strongly 5-HT 2B -preferring relative to 5-HT 2A , indicating that the selectivity for 5-HT 2A in the context of the tetrahydropyridine series may prove challenging.As with the acyclic analogs described in Table 1, this potent 5-HT 2B agonist activity is concerning with respect to the potential to induce pulmonary arterial hypertension (PAH), valvular heart disease (VHD), and related cardiopathies. 32A functional agonist profile at 5-HT 2B , however, does not necessarily guarantee cardiotoxicity, and in fact partial agonists for this receptor have been shown to prevent and treat Sugen-hypoxiainduced PAH in mice. 39Furthermore, signaling bias may play a role in determining the cardiotoxic potential of a given compound; compounds including ropinirole and BW723C86 are not known to induce cardiotoxicity despite being potent functional agonists in the Ca 2+ calcium flux assay. 40,41Further characterization of the present compounds will be needed in order to fully understand any associated risks. 32ncouraged by the high potency of 5-bromo analog 19d (VU6067416), we examined this analog in a battery of in vitro and in vivo pharmacokinetic (PK) assays.VU6067416 (19d) was found to have low predicted hepatic clearance (CL hep ) in human microsomes, with higher turnovers observed for rodent species.Additionally, 19d displayed a high fraction unbound (f u ) in plasma across species, as well as a low predicted P-gp efflux, indicating high potential for brain penetration in human.In a rat iv PK study utilizing cassette dosing, 19d showed moderate plasma clearance (CL p ) and high V ss , with a 2.8 h half-life and a high total brain to plasma ratio (K p ) of 5.4.These parameters, which are summarized in Table 3, are encouraging with respect to the high potential for brain exposure and free drug across species.
19d was found to fully displace radiolabeled racemic 2,5dimethoxy-4-iodomethamphetamine ([ 125 I](±)DOI) in a competition binding experiment for the 5-HT 2A receptor (19d IC 50 = 15 nM), 42 suggesting an orthosteric binding profile.Previous literature has demonstrated the potential for halogenated 5-HT 2A R substrates to form a halogen bond with either or both of the backbone carbonyls of Phe234 5.38 and Val235 5.39 in the 5-HT 2A orthosteric pocket. 43To explore this possibility in the context of the present series, VU6067416 (19d) was docked to an active state of 5-HT 2A R (PDB code 7RAN) 38 using AutoDock VinaXB (Figure 2A). 44While traditional 5-HT 2A R agonist binding interactions with Phe340 6.52 (π−π with indazole) and Asp155 3.32 (salt bridge with amine) are retained, the distance between the bromine and Phe234 5.38 backbone carbonyl oxygen is slightly outside the typical cutoff for halogen bond formation between a bromine and a carbonyl oxygen (3.74 vs 3.37 Å), 45 rendering the occurrence of this phenomenon uncertain in this context.Given the potency of 19d relative to other compounds in the present series and the limitations of rigid docking, though, it is possible that a halogen bond does form.Furthermore, an overlay of the cryo-EM bound pose of 5-HT 2A R agonist (R)-69 38 with the docked pose of 19d indicates a high degree of  similarity between the two conformations, lending credence to this proposed docking mode given the structural similarity of these two agonists (Figure 2B).Definitive structural data, however, are needed to validate this proposed binding mode and any existence of a halogen bond.19d was also docked to an active state of 5-HT 2B R (PDB code 7SRR; orthosteric ligand: LSD) 46 in the same manner (Figure 2C,D).Given that des-bromo analog 19a exhibits nearly identical activity at 5-HT 2B compared to 19d (EC 50 = 16 nM vs 14 nM, respectively), it follows that there were no docking results indicative of a potential halogen bond between the bromine of 19d and the backbone carbonyls of Phe217 5.38 or Met218 5.39 .Rather, the activity at this receptor is likely driven by a combination of ionic interactions, hydrogen bonding, and π−π interactions.
In summary, we report herein a novel series of 5-HT 2 agonists containing an indazole core in place of the traditionally indole-containing tryptamines and show that while many compounds in this series are 5-HT 2A agonists, selectivity relative to the other 5-HT 2 subtypes remains difficult to achieve (and needs to be rigorously profiled, particularly for safety concerns related to 5-HT 2B agonism).
Although nonselective, VU6067416 (19d) is a potent 5-HT 2A agonist (possibly due in part to a halogen bonding interaction with Phe234 5.38 in the 5-HT 2A orthosteric pocket), with favorable PK properties for systemic dosing in rats, and is predicted to be brain-penetrant in human.It is our hope that these results will serve to inform the development of nextgeneration modulators for the 5-HT 2A receptor.

Scheme 4 .
Scheme 4. Synthesis of Indazoles 19a-f, 20, and 21 a Experimental procedures and characterization for new compounds; experimental details for all assays (PDF)■ AUTHOR INFORMATION Corresponding Author Aaron M. Bender − Warren Center for Neuroscience Drug Discovery and Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States;

Table 3 .
In Vitro and in Vivo PK Parameters for VU6067416 (19d) a a See Supporting Information for additional experimental information.